Creutzfeldt-Jakob disease (CJD) is a progressive disorder of the central nervous system and belongs to a class of diseases known as transmissible spongiform encephalopathies, or prion disease. The transmissibility and fatal nature of these diseases necessitates their rapid and accurate diagnosis. The hallmark of these diseases is the accumulation of PrP(Sc), a protease-resistant form of a host-coded glycoprotein. We propose to develop highly sensitive and specific PrP(Sc) detection methods by coupling state-of-the-art immuno-fluorescence techniques with novel fluorescence spectroscopic methods. We have evaluated the use of multi-spectral ultraviolet fluorescent spectroscopy (MUFS) as a means of detecting and distinguishing between different forms of PrP(Sc). Spectroscopic measurements of fluorescence from untreated and proteinase K (PK)-treated PrP(Sc), purified from 263K scrapie strain-infected hamster brains and ME7 scrapie strain-infected mouse brains, were performed. Spectra of untreated and PK-treated PrP(Sc) samples for 263K and ME7 appeared qualitatively different. The identification and discrimination of PrP(Sc) was possible based on these spectral signatures, calculations of their fluorescence cross sections and determination of the orthogonal differences. Furthermore, MUFS was successfully used within an optically dense medium spiked with sodium salicylate. These results indicate that it is possible to detect and discriminate between compounds or proteins in optically dense media, such as undiluted blood plasia, by this method. This technique has the potential not only for the detection of PrP(Sc) in optically dense media, but also for the ability to distinguish between different forms of the prion protein. Furthermore, in combination with multi-site directed, fluorophore-tagged monoclonal antibodies, this method will prove to be an extremely powerful tool for the analysis of PrP(Sc) and the diagnosis not only of CJD, but other prion diseases.